This invention relates to the field of laxatives, treatments to lower blood serum cholesterol and low-calorie food thickeners and fat substitutes. In particular, the invention relates to unfermented gel-forming polysaccharides from psyllium seed husks and methods for their isolation.
Various scientific and scholarly articles are referred to in parentheses throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
The seed husks of psyllium (Plantago ovata, also known as ispaghula) are commonly used as a laxative and to promote regular bowel function. Psyllium seed husks promote Taxation partially by increasing the mass and moisture content of the stool (Marteau et al., 1994, Gut 35:1747-1752). Additionally, the excreta of animals and humans fed diets containing psyllium seed husk is gelatinous. This gelatinous property contributes to laxative properties of psyllium seed husks by decreasing friction in the gut. Observed increases in fecal mass and water retention also have been attributed to this gelatinous material (Marteau et al., 1994, supra). The gel is composed largely of unfermented psyllium polysaccharides (Cabotaje et al., 1994, 1302-1307).
Currently used preparations of psyllium seed husks have certain disadvantages. Laxative preparations of psyllium seed husks are generally composed of ground husk and have coarse and unpleasant mouthfeel when administered in drinks. Psyllium seed husks have been incorporated into cookies, crackers and similar products; however, these products have a tendency to begin to gel unpleasantly in the mouth. More significantly, though, psyllium seed husks can swell in the esophagus, producing an esophageal obstruction that can cause choking. For this reason, psyllium seed husk preparations are not recommended for ingestion by persons who may have difficulty swallowing (e.g., elderly persons). Finally, the recommended daily dose of psyllium husk of 3.5-11 g per day is inconvenient to ingest in any form. What is needed is a form of psyllium husk that is convenient and pleasant to use.
Psyllium seed husk has many of the properties of soluble dietary fiber sources. Commonly used sources of soluble dietary fiber include pectin, gums and oat bran. Soluble dietary fiber (SDF) has many uses in food and medicinal preparations. Soluble fibers are components of minimally processed food sources such as oats, oat bran and barley, or are available as concentrates, such as gums, pectins and mucilages. Gums and mucilages are carbohydrate polymers that are generally isolated from plant sources. Mucilages in particular produce slippery or gelatinous solutions in water. Pectins are polymeric chains of partially methylated galacturonic acids that also possess the ability to form a gel in water. Most soluble fibers are rapidly and completely fermented and have no Taxation properties.
Sources of soluble dietary fiber that are also viscous lower serum cholesterol in animals and humans (Marlett, 1997, pp. 109-121, Dietary Fiber and Health, Plenum Press, New York, ed. Kritchevsky and Bonfield). The viscosity of the SDF, rather than its fermentation in the gastrointestinal tract, is key to its hypocholesterolemic action (Marlett et al., 1994, Hepatology 20:1450-1457). Viscosity in the lumen of the lower small intestine interferes with the absorption of bile acids and more bile acids are lost through the stool. Blood cholesterol is thought to be lowered primarily because it is being used in the liver to synthesize more bile acids to replace those lost. The synthesis of bile acids in the liver accounts for 40 to 50% of the daily elimination of cholesterol from the blood. However, the addition of one source of soluble fiber, oat bran, to the diet also increases the proportion of deoxycholic acid in the bile acid pool, which decreases the absorption of exogenous dietary cholesterol. Supplementing the diet with psyllium husk also increases the excretion of bile acids by about 50% (Gelissen et al., 1994, Am. J. Clin. Nutr. 59:395-400).
Soluble dietary fiber concentrates are also often used as thickeners and low calorie fat substitutes in the food industry because of their hydrocolloidal properties (Ward, 1997, Cereal Foods World, 42:386-390). Low-viscosity gums such as gum acacia have both hydrophilic and lipophilic properties that make them ideal as emulsifiers, surfactants and stabilizers. Pectins and mucilages have gel-forming properties that made them ideal thickeners of food products. Pectins are traditionally extracted from apple and citrus fruits. Commonly used mucilages are generally extracted from seaweed and include carrageenan, agar and alginate. A fat substitute can be made by combining gum with mucilage and/or pectin to create a compound with the emulsifying properties and smoothness of a fat.
The present invention provides the gel-forming component of psyllium seed husks in a purified form. This gel fraction provides the Taxation and hypocholesterolemic effects of intact psyllium seed husks, but is in a form that is easily administrable as a tablet, capsule or liquid, without certain unpleasant or unsafe qualities associated with the use of intact psyllium seed husks. The gel fraction also has utility in treatment of other intestinal abnormalities and maintaining normal bowel function, and as a food thickener and fat replacement.
According to one aspect of the invention, a gel-forming fraction of psyllium seed husks that survives microbial fermentation upon passage through a monogastric mammalian digestive tract is provided. Among other components, the gel fraction comprises predominantly xylose and arabinose in a dry weight ratio of at least about (preferably about 3.5). The fraction comprises notably limited amounts of other sugars, e.g., about 2.5%-13.5% total of rhamnose, galactose, glucose and uronic acids. More specifically, the gel-forming fraction has the following sugar composition, expressed as a percentage of total sugars:
between about 0.5% and 4% rhamnose;
between about 19% and 22% arabinose;
between about 68% and 76% xylose;
between about 0% and 0.5% mannose;
between about 1% and 2% galactose
between about 0% and 1% glucose; and
between about 1% and 6% uronic acids
Upon further purification, the gel-forming fraction becomes even more depleted in rhamnose, glucose and uronic acids.
The gel-forming fraction is also highly viscous, a 0.2% concentration in formamide having an apparent viscosity of at least 500 sec, preferably 750 sec, and most preferably 850 sec. The fraction is soluble in a dilute alkaline solution and forms a gel upon acidification of the solution to a final pH of about 4.5.
According to another aspect of the invention, in a preferred method of obtaining the psyllium seed husk gel-forming fraction, a separate carbohydrate fraction is also obtained. This fraction is soluble in the dilute alkaline solution and remains soluble upon acidification of the solution to a pH of about 4.5. This fraction is comprised of xylose and arabinose in a ratio of at least about 4:1 and further comprises at least about 12% (by weight) rhamnose and at least about 15% (by weight) uronic acids.
According to another aspect of the invention, a method of fractionating psyllium seed husks to obtain a gel-forming fraction and an additional carbohydrate fraction is provided. The method comprises: (a) mixing the husks, in the presence of a chemical reducing agent, in an aqueous alkaline solution comprising between about 0.15 and about 1.0 M (preferably 0.15-0.5 M, more preferably 0.15-0.4 M, even more preferably 0.15-0.3 M and most preferably 0.15-0.2 M) hydroxyl ions, thereby fractionating the husks into an alkali soluble fraction and an alkali-insoluble fraction; (b) removing the alkali insoluble fraction; (c) acidifying the alkali soluble fraction to a pH of between about 3 and about 6 (preferably between about 4 and about 5, most preferably about 4.5), which results in the gelation of the gel-forming fraction; and (d) separating the gel fraction, e.g., by centrifugation, from the additional carbohydrate fraction contained in the acidified solution. In preferred embodiments, the method further comprises washing the gel fraction with an aqueous or buffered solution and desiccating the washed gel fraction.
In another aspect of the invention, a gel-forming fraction from psyllium seed husks is provided, which is produced by the aforementioned procedure. The additional carbohydrate fraction is also provided in this aspect of the invention.
According to another aspect of the invention, alternative methods are provided for obtaining a polysaccharide fraction that contains the aforementioned gel-forming fraction. Such a fraction is obtained by solvent extraction using formamide, dimethylsulfoxide or 4-methylmorpholine N-oxide (50% solution in water). The solvent-treated material is centrifuged to recover the soluble materials that are then poured into ethanol to achieve a concentration of 80% ethanol. The precipitate that forms is similar in composition to the alkali-soluble gel-forming fraction.
According to another aspect of the invention, pharmaceutical preparations for treatment of constipation or other intestinal abnormalities, or for lowering serum cholesterol levels in a patient are provided. These preparations are formulated to contain effective dosages of the psyllium seed husk gel-forming fraction of the invention. Methods of treating patients for these various conditions are also provided, which comprise administering the pharmaceutical preparations of the invention.
Other features and advantages of the present invention will become apparent from the detailed description and examples that follow.
The present invention provides a highly polymerized gel-forming fraction from psyllium seed husks that has great utility in the treatment and prevention of constipation and as a hypocholesterolemic agent. This gel fraction remains substantially unfermented during transit through the gastrointestinal tract and promotes laxation through a variety of means, including increasing moisture content and overall mass of stool and imparting to the stool a slippery characteristic that facilitates ease of passage of stool (Example 6).
The gel-forming fraction from psyllium seed husks has also been demonstrated as a hypocholesterolemic agent. Accordingly, the gel-forming fraction may be used alone or in combination with other active substances as a therapeutic treatment to lower serum cholesterol.
Stool from rats and humans fed psyllium seed husks are sometimes gelatinous (Cabotaje et al., 1994, supra; Example 6). However, attempts to purify and characterize a gel-forming material (Kennedy et al., Carbohydrate Res. 75:265-274, 1979; Sandhu et al., Carbohydrate Res. 93:247-259, 1981) have not been successful and none has provided the highly purified gel-forming fraction having the features described in accordance with the present invention. The method of fractionating psyllium seed husks developed in accordance with the invention has led to the unexpected discovery, contrary to published reports (e.g., Kennedy et al., 1979, supra), that a gelatinous, alkali-soluble fraction of psyllium seed husks can be further fractionated to form a highly viscous gel fraction (referred to herein as xe2x80x9cFraction Bxe2x80x9d) and a second carbohydrate fraction with distinctive compositional features, as described in greater detail below (referred to herein as xe2x80x9cFraction Cxe2x80x9d). Both the viscous, gel-forming Fraction B and the additional Fraction C are soluble in a variety of substances, including dilute and concentrated alkali, formamide, dimethysulfoxide and 4-methylmorpholine N-oxide (50% aqueous solution); consequently, these two fractions together can be isolated on the basis of these solubility characteristics. However, the further separation of the fractions has been either unsuccessful (e.g., a strong alkali-extracted gel-forming fraction was unable to be further separated by Kennedy et al., 1979; supra) or has remained unexplored. The present inventors have discovered that, using a suitable first extraction procedure to obtain a product comprising fractions B and C together, that the fractions can be separated by acidification of a solution containing a mixture of the fractions. Fraction B is concentrated for separation from the acidified mixture by centrifugation, whereas Fraction C remains soluble in the acid.
Description of Carbohydrate Polymers
The viscous, gel-forming psyllium seed husk fraction of the invention (Fraction B) is comprised primarily of xylose and arabinose. In a preferred embodiment, the gel-forming fraction has at least 50% xylose and arabinose by weight, in a more preferred embodiment at least 75% xylose and arabinose by weight, and in a most preferred embodiment at least 85% xylose and arabinose by weight. The gel-forming fraction has an apparent viscosity, as determined in the method of Example 3, of at least 500 sec, in a more preferred embodiment of at least 750 sec, and in a most preferred embodiment of at least 350 sec. The gel-forming fraction is furthermore particularly deficient in rhamnose, galactose and uronic acids, as compared to xylose. In a preferred embodiment, the ratio of weights of xylose to rhamnose is more than 50, in a more preferred embodiment the ratio is more than 60, and in a most preferred embodiment, the ratio is more than 65. In a preferred embodiment, the ratio of weights of xylose to galactose is more than 25, in a more preferred embodiment the ratio is more than 35, and in a most preferred embodiment, the ratio is more than 42. In a preferred embodiment, the ratio of weights of xylose to uronic acid is more than 15, in a more preferred embodiment the ratio is more than 25, and in a most preferred embodiment, the ratio is more than 35. In a preferred embodiment, the ratio of weights of xylose to arabinose of Fraction B is between 2.5 and 4.5, in a more preferred embodiment the ratio is between 3.0 and 4.0, and in a most preferred embodiment, the ratio is between 3.25 and 3.75.
The acid-soluble psyllium seed husk fraction (Fraction C) is also high in xylose and arabinose. In a preferred embodiment, the acid-soluble fraction has at least 25% xylose and arabinose by weight, in a more preferred embodiment at least 40% xylose and arabinose by weight, and in a most preferred embodiment at least 45% xylose and arabinose by weight. Though Fraction C has an apparent viscosity similar to that of fraction B, it does not have the gel-forming property of Fraction B. Fraction C is furthermore particularly enriched in rhamnose, galactose and uronic acids, as compared to xylose. In a preferred embodiment, the ratio of weights of xylose to rhamnose is less than 6.0, in a more preferred embodiment the ratio is less than 4.5, and in a most preferred embodiment, the ratio is less than 3.0. In a preferred embodiment, the ratio of weights of xylose to galactose is less than 40, in a more preferred embodiment the ratio is less than 30, and in a most preferred embodiment, the ratio is less than 25. In a preferred embodiment, the ratio of weights of xylose to uronic acid is less than 30, in a more preferred embodiment the ratio is less than 10, and in a most preferred embodiment, the ratio is less than 5.0 In a preferred embodiment, the ratio of weights of xylose to arabinose of Fraction C is more than 3.0, in a more preferred embodiment the ratio is more than 4.0, and in a most preferred embodiment, the ratio is more than 4.5.
Preparation of Psyllium Seed Husk Fractions
The present invention also provides methods of fractionating psyllium seed husks to yield the purified and separated fractions described above. In its most basic form, the method has the following steps:
1. Suspend psyllium seed husks in a dilute alkaline aqueous solution (preferably 0.15-0.2 M, hydroxyl ions) containing a reducing agent, in which portions of the husk material will dissolve, while a certain portion remains insoluble;
2. Remove the alkaline-insoluble material (referred to herein as xe2x80x9cFraction Axe2x80x9d), e.g., by centrifugation;
3. Acidify the alkali-soluble fraction of step one to a pH of between 3 and 6, preferably 4.5, to yield a gel (Fraction B) and an acid-soluble fraction (Fraction C); and
4. Separate the gel from the acidified solution, e.g., by centrifugation.
One example of this method is taught in Example 1. Many variations exist to the method that will not substantially change the product isolated. These are described in detail below.
The alkaline solubilization step has several variations. The method taught in Example 1 has improved this solubilization over that found in the prior art. Previous alkaline solubilizations of psyllium seed husk polysaccharides utilized concentrated solutions of base (i.e. 1.2 M NaOH, Kennedy et al., 1979, supra) without a reducing agent. Recognizing the harsh nature of this treatment and its partial degradation of polysaccharide chains in the gel-forming fraction, the inventors have demonstrated that a gel-forming fraction can be obtained, presumably in a form more suitable for further fractionation, using a much less concentrated alkaline solution and a suitable chemical reducing agent, such as borohydride. Though up to 4 N alkaline solution can be utilized, the concentration of base in the alkaline solubilization is preferably at least 0.15 N and not more than 1.0 N; in a more preferred embodiment, at least 0.15 N and not more than 0.5 N; and in the most preferred embodiment, at least 0.15 N and not more than 0.2-0.3 N. Any standard base can be used in the alkaline extraction, including, but not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide and tetramethyl ammonium hydroxide.
A chemical reducing agent, such as borohydride, should be added to the alkaline solubilization step to minimize base-catalyzed depolymerization. In Example 1, a concentration of 1 g/L of sodium borohydride is used, but effective concentrations range from about 50 mg/L to 10 g/L. In a preferred embodiment, the sodium borohydride concentration is at least 100 mg/L and not more than 4 g/L, in a more preferred embodiment at least 500 mg/L and not more than 2 g/L, and in a most preferred embodiment, at least 800 mg/L and not more than 1.2 g/L. Other forms of borohydride also are suitable for use in this step, including but not limited to, lithium borohydride, potassium borohydride and sodium cyanoborohydride.
The degree of initial processing of psyllium seed husks may alter the alkaline solubilization in ways that will be well known to those skilled in the art. It is important that the husk material be processed so that it is in small pieces, in order to allow the viscous polysaccharides to be easily separated from the insoluble and fibrous materials of the cell walls. In Example 1, the psyllium seed husks are milled, but any process that pulverizes the plant material can be used, and these processes are well known in the art.
The ratio of seed husk material to alkaline solution can be important for efficient solubilization of the polysaccharide fractions. In Example 1, a ratio of 2 g psyllium seed husk is added to 400 ml of alkaline solution, but this ratio can be varied without appreciably affecting the solubilization. For instance, the ratio can be varied so as to add as little as about 0.1 g seed husks or as much as about 4 g seed husks to a 400 ml alkaline solution. Additionally, the time of solubilization can be varied to optimize the procedure (0.5 hr-24 hr) at a range of temperatures (4-50xc2x0 C).
Step two of the method of the invention requires that the alkaline insoluble materials be separated from the alkaline soluble materials. In Example 1, centrifugation is employed to accomplish this objective. However, numerous variations and other procedures may be substituted without substantially changing the soluble materials isolated. One skilled in the art will know how to alter the time and force of the centrifugation to adapt the separation to different centrifuge rotors, plant materials and alkaline solutions. Other methods that will accomplish this separation are well known in the art. Some of these methods will be better suited to large scale use of the method of the invention. Separation methods of interest include, but are not limited to, flow-through centrifugation or filtration (with agitation). Example 1 further teaches washing the insoluble materials with the alkaline solution and re-separation to improve the yield of the alkaline soluble materials. This washing step is optional but can be used to advantage to improve yield.
Step three requires that the alkaline soluble materials of step two be subjected to acidification. In Example 1 this is accomplished by adding glacial acetic acid to the combined alkaline soluble materials until the pH is adjusted to 4.5. The range of pH used for this acid solubilization can be varied without substantial effect on the products. In a preferred embodiment, the pH is between 3 and 6, in a more preferred embodiment, the pH is between 4 and 5, and in a most preferred embodiment, the pH is about 4.5 as described in Example 1. The choice of acid is also subject to variation. Examples of acids suitable for use in this step are acetic, hydrochloric, sulfuric, oxalic, trichloroacetic and trifluoroacetic acids, among others. Here as in step one, the duration, temperature, etc. of the solubilization can be varied, but preferably is carried out at ambient temperature for about 2 hours.
Step four requires that the acid insoluble gel-like material (Fraction B) be separated from the acid soluble materials (Fraction C). Centrifugation is typically employed to accomplish this separation. An optional washing of the insoluble gel mass (e.g., with water, buffer or other suitable solvent) may also be performed to improve the efficiency of the separation.
Preparing Fraction B and Fraction C for storage and/or use may employ several procedures. The polysaccharide preparations of Fractions B and C may be used or stored hydrated. If stored in a hydrated form, preservatives or bacteriostatic agents may be added. Drying the polysaccharide preparations is particularly advantageous for use or storage. In a preferred embodiment, Fraction B and Fraction C are desiccated by treatment with 95% ethanol, washing with diethyl ether and drying. The fractions also may be desiccated with other solvents, such as methanol, acetone or isopropyl alcohol. Any standard dehydration method (e.g., evaporation, lyophilization) may be used to dry the fractions, provided the temperature is maintained at less than about 40xc2x0 C.
III. Uses of Psyllium Seed Husk Fractions
The psyllium seed husk fractions of the invention have uses as therapeutic treatments. In this regard, the viscous, gel-forming fraction, Fraction B, has been demonstrated effective in promoting Taxation and also as a hypocholesterolemic agent. This material can be used alone or in combination with other active substances in therapeutic or prophylactic preparations for constipation, diarrhea and/or high serum cholesterol. Such preparations can incorporate the gel-forming fraction in pills, capsules or liquids to be administered by mouth. In a preferred embodiment, a dried form of the gel is formulated for convenient administration as a pill or capsule. The gel re-hydrates upon ingestion. The gel-forming preparations can additionally be incorporated into food products. Since the active polysaccharides have been isolated away from the other plant cell components by the method of the invention, they will not have the unpleasant mouthfeel or the necessity to administer large dosages associated with the psyllium seed husk preparations currently in use.
The unfermented gel-forming polysaccharide from psyllium seed husks is well-known for its laxative effects on the monogastric mammalian digestive tract. For an adult human, a suitable dosage of the gel-forming fraction in dry form is about 2 g, one to three times a day, to maintain bowel regularity and as a treatment for constipation.
As described in Example 4, the gel-forming fraction of psyllium seed husks has been demonstrated as a hypocholesterolemic agent. Accordingly, this fraction also may be used alone or in combination with other active substances as a therapeutic treatment to lower serum cholesterol. For an adult human, a suitable dosage of Fraction B in dry form is from about 3 g to about 7 g daily. The psyllium husk Fractions B and C of the invention can additionally be used as food additives. They may be used as thickeners, gel-formers and bulking agents in prepared foods. They may also be combined with other food additives to make fat mimetic systems. Because the polysaccharide preparations of the invention are partially non-digested, they will additionally be low calorie, serum cholestrol-lowering and laxation-promoting. The polysaccharide preparations of Fractions B and C can be used in many of the food products where gums and mucilages are currently used.
The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.